Methylotrophic yeasts are those yeasts that are able to utilize methanol as a sole source of carbon and energy. Species of yeasts that have the biochemical pathways necessary for methanol utilization are classified in four genera, Hansenula, Pichia, Candida, and Torulopsis. These genera are somewhat artificial, having been based on cell morphology and growth characteristics, and do not reflect close genetic relationships (Billon-Grand, Mycotaxon 35:201-204, 1989; Kurtzman, Mycologia 84:72-76, 1992). Furthermore, not all species within these genera are capable of utilizing methanol as a source of carbon and energy. As a consequence of this classification, there are great differences in physiology and metabolism between individual species of a genus.
Methylotrophic yeasts are attractive candidates for use in recombinant protein production systems. Some methylotrophic yeasts have been shown to grow rapidly to high biomass on minimal defined media. Certain genes of methylotrophic yeasts are tightly regulated and highly expressed under induced or de-repressed conditions, suggesting that promoters of these genes might be useful for producing polypeptides of commercial value. See, for example, Faber et al., Yeast 11:1331, 1995; Romanos et al., Yeast 8:423, 1992; and Cregg et al., Bio/Technology 11:905, 1993.
Development of methylotrophic yeasts as hosts for use in recombinant production systems has been slow, due in part to a lack of suitable materials (e.g., promoters, selectable markers, and mutant host cells) and methods (e.g., transformation techniques). The most highly developed methylotrophic host systems utilize Pichia pastoris and Hansenula polymorpha (Faber et al., Curr. Genet. 25:305-310, 1994; Cregg et al., ibid.; Romanos et al., ibid.; U.S. Pat. No. 4,855,242; U.S. Pat. No. 4,857,467; U.S. Pat. No. 4,879,23 1; and U.S. Pat. No. 4,929,555).
Minimization of spurious proteolysis of recombinant proteins generated under high cell density fermentation conditions is highly desirable. In yeast, the major store of proteolytic activity is located within the lumen of the vacuolar compartment (Jones, Methods Enzymol. 194:428-453, 1991). These proteases are released into the fermentation broth by spontaneous and inevitable cell lysis and are further liberated during cell breakage that is required to release intracellulary produced proteins in laboratory or industrial production. Although vacuolar proteases are required for several developmental transitions in the life cycle of yeast cells (e.g., sporulation), they are dispensible for vegetative growth. The majority of vacuolar proteases are synthesized and transported through the secretory pathway as enzymatically inactive zymogens (Klionsky et al., Microbiol. Rev. 54:266-292, 1990; Raymond et al., Int. Rev. Cytol. 139:59-120, 1992). They are proteolytically activated by the combined action of proteinase A, the product of the PEP4 gene, and proteinase B, the product of the PRB1 gene; hence pep4 prb1 mutants are generally considered to be fully protease-deficient (Jones, ibid.). Although protease-deficient strains of several species of yeast have been described (e.g., Gleeson et al., U.S. Pat. No. 5,324,660; Jones, Methods Enzymol. 194:428-453, 1991; Fleer et al., WO 94/00579), protease-deficient strains of P. methanolica have not been available and methods for the generation of such strains have not heretofore been available.
There remains a need in the art for strains and techniques that will facilitate the commercial development of additional species of methylotrophic yeasts, including the use of Pichia methanolica to produce polypeptides of economic importance. The present invention provides such strains and techniques, as well as other, related advantages.